Proteolytic Regulation of Activated STAT6 by Calpains1
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免疫学杂志 2005年第5期
Abstract
The transcription factor STAT6 plays an important role in cell responses to IL-4. Its activation is tightly regulated. STAT6 phosphorylation is associated with JAKs, whereas dephosphorylation is associated with specific phosphatases. Several studies indicate that proteases can also regulate STAT6. The aim of this study was to investigate the nature of these proteases in mouse T cell lines. We found that STAT6 was degraded in cell extracts by calcium-dependent proteases. This degradation was specifically prevented by calpain inhibitors, suggesting that STAT6 was a target for these proteases. This was supported by the cleavage of STAT6 by recombinant calpains. The proteolytic regulation of STAT6 was more complex in vivo. Calcium signaling was not sufficient to induce STAT6 degradation. However, treatment of IL-4-stimulated cells with calcium ionophores resulted in the absence of phosphorylated STAT6. This effect correlated with the loss of STAT6 protein and was prevented by calpain inhibitors. Cytoplasmic calpains seemed to be responsible for STAT6 degradation. Calpains can target signaling proteins; in this study we found that they can negatively regulate activated STAT6.
Introduction
Interleukin-4 is a cytokine with a wide spectrum of biological activities (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). IL-4 is mainly produced by T cells, mast cells, basophils, and eosinophils, but its effects are not limited to the immune system (2). IL-4 can regulate proliferation, differentiation, and apoptosis in multiple cell types of hemopoietic and nonhemopoietic origin (3, 4, 5). One of the most important effects of IL-4 is the regulation of T cell differentiation. IL-4 promotes the formation of Th2 cells while inhibiting Th1 (6). IL-4-driven Th2 cells can be protective in rheumatoid arthritis (7) and helminthic infections (8), but conversely, they can have an important role in allergic disease progression (9).
Responses to IL-4 are mediated by a cell surface receptor complex expressed in most cell types (2). This receptor consists of two subunits, the IL-4R -chain (IL-4R) and the common -chain (10). The binding of IL-4 to its receptor provokes the activation of JAK tyrosine kinases and the phosphorylation of tyrosine residues within the cytoplasmic tail of the receptor (11). Thus, domains of the IL-4R containing phosphorylated tyrosines can recruit intracellular signaling molecules. Among them, the transcription factor STAT6 plays an important role, as demonstrated by the similar phenotype of mice lacking the IL-4R and those lacking STAT6 (12).
The mechanism proposed for STAT6 activation is similar to that of other STATs (11, 12, 13, 14, 15). STAT6 binds through its Src homology 2 domain to specific phosphotyrosine residues within the IL-4R (15). In this complex, STAT6 is also phosphorylated at tyrosine residues (14). Subsequently, STAT6 leaves the receptor, dimerizes, and migrates to the nucleus, where it binds to consensus sequences in the promoter of genes (2, 13). It is believed that STAT6 is tightly regulated by kinases, phosphatases, and proteases. Since its discovery, the activation of STAT6 was associated with JAKs (11, 14). However, other tyrosine kinases, such as Src, can participate in the earlier events that lead to STAT6 activation (16). The inhibition of STAT6 is also strongly regulated, because in the absence of IL-4, STAT6 is quickly deactivated. Thus, phosphatases, by dephosphorylating STAT6 (17, 18), and suppressor of cytokine signaling, by blocking the kinase activity associated with the receptor (19), are inhibitors of STAT6.
Recent findings indicate that proteases can also be involved in the regulation of STAT6 (17, 20, 21, 22). Two research groups have reported that the cleavage of STAT6 by nuclear proteases can be an important event in the regulation of transcription in mast cells (20, 21). The nature of these proteases is still under investigation, although the data reported suggest that they may belong to the family of serine proteases. Calpains have been found to be able to cleave STAT6 (22) and other STATs (23), although their role in signaling is still unknown. The proteasome has also been involved in the inhibition of STAT6, although by an indirect mechanism (17, 19). Given the importance that proteases may have in the regulation of STATs, we investigated the nature of STAT6 proteases and their role in signaling. We found that in vitro, STAT6 was proteolytically degraded by calpains in a calcium-dependent manner. However, calcium signaling was not enough to promote in vivo STAT6 degradation. Our results suggest that only IL-4-activated STAT6 was an in vivo substrate for calpain proteases.
Materials and Methods
TCells and reagents
The murine T cell hybridoma A1.1 was maintained in RPMI 1640 culture medium with glutamine, penicillin, streptomycin, and 10% FCS (complete medium). A23187, N-acetyl-Leu-Leu-Nle-CHO (ALLN),3 N-acetyl-Leu-Leu-Met-CHO, calpastatin peptide, recombinant rat m-calpain, and purified μ-calpain from human erythrocytes were purchased from Calbiochem. The caspase inhibitors Boc-Asp-fluoromethylketone and z-Val-Ala-Asp-fluoromethylketone (z-VAD) were obtained from Alexis. RC20 anti-phosphotyrosine Ab was purchased from Transduction Laboratories, and M20 and M200 anti-STAT6 Abs were obtained from Santa Cruz Biotechnology. Cytokines were purchased from R&D Systems. The rest of the reagents used were purchased from Sigma-Aldrich.
Protease assays in cell extracts
After culture, cells were centrifuged and lysed with lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 0.5% Brij 35, and 0.1% Nonidet P-40) without inhibitors. Lysates were then clarified by centrifugation at 20,000 x g for 10 min. The soluble fraction was incubated as indicated to determine STAT6 protease activity. The reaction was stopped by adding 2x SDS sample buffer, and samples were separated on a 7.5% SDS-polyacrylamide gel before transfer to a polyvinylidene difluoride membrane. Membranes were then probed with M20 or M200 anti-STAT6 Abs. The bound Ab was detected using ECL (Pierce).
Immunoprecipitation and immunoblotting
These experiments were performed as we have previously described (16). After the indicated culture conditions, cell pellets were lysed with lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 50 mM sodium fluoride, 10 mM pyrophosphate, 1 mM PMSF, and protease inhibitor mixture) and clarified by centrifugation. The soluble fraction was incubated with anti-STAT6 Ab, followed by protein G-agarose. The washed precipitates were analyzed by Western blot as described above.
Preparation of cytoplasmic and nuclear extracts
Nuclear and cytoplasmic extracts were obtained as previously described with some modification (18, 24). After culture, cells were washed with buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 1,5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, and protease inhibitor mixture). Then cells were incubated for 20 min on ice with buffer A supplemented with 0.1% Nonidet P-40. Samples were centrifuged at 14,000 x g for 30 s. The supernatant containing the cytoplasmic extract was retained. The pellet containing nuclei was washed twice with buffer A, then resuspended in buffer B (20 mM HEPES (pH 7.9), 420 mM NaCl, 1,5 mM MgCl2, 1 mM EDTA, 0.5% Nonidet P-40, 0.5 mM DTT, and protease inhibitor mixture). After 10 min of incubation, samples were centrifuged at 14,000 x g for 10 min. The supernatant containing the nuclear extract was diluted with buffer B without NaCl to a final concentration of 150 mM NaCl. STAT6 was then precipitated and analyzed in cytoplasmic and nuclear extracts as described above.
To investigate STAT6 protease activity, cytoplasmic and nuclear extracts were obtained from A23187-stimulated cells as described above, but all procedures were performed in the absence of EDTA and protease inhibitors. In parallel, STAT6 was precipitated from IL-4-stimulated cells, and immunocomplexes were extensively washed in protease reaction buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 0.5% Brij 35, 0.1% Nonidet P-40, and 0.5 mM DTT). Then immunoprecipitates containing STAT6 were incubated with cytoplasmic and nuclear extracts in reaction buffer. After that, immunoprecipitates containing STAT6 were analyzed by Western blot as described above. The relative densities of STAT6 bands were analyzed in the total STAT6 protein blots using Science Lab software from Fuji Photo Film.
Results
Degradation of STAT6 by calpains
Several groups have reported that proteases can participate in the regulation of STAT6, although the mechanisms are not fully understood (17, 20, 21, 22). In this study we investigated the nature of these proteases. As a first approach, we analyzed the stability of STAT6 in cell extracts obtained in the absence of protease inhibitors (Fig. 1A). Incubation of these extracts at 20 and 37°C resulted in no significant loss of STAT6 protein compared with samples kept on ice. The lack of STAT6 degradation could be due to the absence in the preparations of cofactors that are necessary for the enzymatic activity of proteases. Because cations are protease cofactors, we investigated whether they influenced the stability of STAT6 (Fig. 1B). The addition of Zn2+, Mg2+, and Mn2+ to the extracts had little effect on STAT6 protein levels. In contrast, the addition of Ca2+ provoked the complete loss of STAT6, indicating that it was a substrate for calcium-dependent proteases. To identify these proteases, these cell extracts were incubated with calcium in the presence of known protease inhibitors (Fig. 1C). Among all inhibitors tested, only those known to inhibit calpains were able to block the calcium-dependent STAT6 degradation. Thus, EDTA, ALLN, and calpastatin blocked the degradation of STAT6, whereas inhibitors of caspase (Boc-Asp-fluoromethylketone and z-VAD) and serine protease (N-p-tosyl-L-phenylalanine chloromethylketone) had no effect (Fig. 1C). These data strongly suggested that STAT6 was an in vitro substrate for calpain proteases. This was confirmed by the fact that STAT6 was cleaved by recombinant m-calpain (Fig. 2). To perform these experiments, STAT6 was immunoprecipitated from untreated A1.1 cells to avoid potential interfering factors that could be present in the cellular extracts. Thus, the incubation of precipitated STAT6 with recombinant m-calpain resulted in the degradation of STAT6 by a calcium-dependent mechanism (Fig. 2A, left). This effect was also prevented by calpain inhibitors. Similar results were
found when immunoprecipitated STAT6 was incubated with purified human m-calpain (Fig. 2A, right). To detect STAT6, we used the M200 anti-STAT6 Ab that recognizes an internal domain and can detect shorter products of STAT6 proteolysis (22). However, we did not observe shorter STAT6 fragments even though we carefully performed dose- and time-response experiments (Fig. 2B). Thus, immunoprecipitated STAT6 was quickly degraded by different doses of recombinant m-calpain in the presence of calcium. The loss of STAT6 was not accompanied by the appearance of shorter fragments. It could be possible that the cleavage of STAT6 by calpains would produce fragments too small to be detected. These findings were consistent with the presence of multiple potential PEST domains in STAT6 that can be cleaved by calpains (25). Similar results were obtained with M20 Ab that recognizes a STAT6 C-terminal domain (data not shown). Taken together, these data indicated that STAT6 was a substrate to be proteolytically degraded by calpain in vitro.
FIGURE 1. STAT6 is proteolytically degraded by calcium-dependent proteases. A, Cells extracts obtained from A1.1 cells in the absence of protease inhibitors were kept at 4°C or incubated at 20 and 37°C for 30 min. Then samples were separated by SDS-PAGE and immunoblotted with M200 anti-STAT6 Ab. B, Cell extracts were supplemented with 3 mM of the indicated cation and incubated for 30 min at 20°C. STAT6 was analyzed as described above. C, Cell extracts were left untreated (Untreat.) or were incubated for 10 min with nothing or with 10 mM EDTA, 5 μM calpastatin (CAST), 20 μM of the calpain inhibitors N-acetyl-Leu-Leu-Met-CHO and ALLN, 20 μM of the caspase inhibitor z-VAD, and 40 μM of the serine protease inhibitor N-p-tosyl-L-phenylalanine chloromethylketone. Then 3 mM CaCl2 was added, and samples were incubated for 20 min at 20°C. STAT6 was analyzed as described in A. These experiments were performed at least three times.
FIGURE 2. Degradation of STAT6 by recombinant calpains. A, STAT6 was immunoprecipitated from unstimulated A.1.1 cells. Then it was incubated with recombinant m-calpain (left) and purified human μ-calpain (right) for 10 min in the presence of nothing, EDTA, or the indicated protease inhibitors. To start the reactions, 3 mM CaCl2 was added, and samples were incubated for 20 min at 20°C. As a control, STAT6 was incubated with calpain in the absence of CaCl2 (NoCa). STAT6 was detected by Western blotting with M200 anti-STAT6 Ab. B, Immunoprecipitated STAT6 was incubated with the indicated amount of recombinant m-calpain before the addition of 3 mM CaCl2. Then samples were incubated for 2 and 4 min at 20°C. As controls, samples were kept on ice for 4 min (untreat). STAT6 was analyzed as described above. These experiments were performed at least three times.
Proteolytic regulation of STAT6 by calcium signaling
Calpains are calcium-dependent proteases that can cleave intracellular signaling proteins (26). Calcium ionophores increase intracellular calcium levels, provoking the activation of calpains (27, 28). However, stimulation of cells with the calcium ionophore A23187 did not induce STAT6 degradation in cells (Fig. 3A), suggesting that STAT6 could not be an in vivo calpain substrate. Alternatively, it could be possible that calcium signaling was not enough to promote STAT6 degradation. This hypothesis was based on the fact that some substrates require tyrosine phosphorylation to be cleaved by calpains (29, 30). Therefore, we investigated whether STAT6 phosphorylation by IL-4 could favor its degradation by calpain (Fig. 3B). IL-4 induced a rapid and strong phosphorylation of STAT6 that decayed over time, as previously demonstrated (17, 18). Thus, the levels of STAT6 phosphorylation were higher after 30 min than after 2 h of stimulation with IL-4. Treatment of A1.1 cells with calcium ionophore for 30 min did not
affect the ability of IL-4 to induce STAT6 phosphorylation. At this time, the levels of STAT6 protein also remained similar under all treatments. In contrast to that at 30 min, the phosphorylation of STAT6 induced by IL-4 was completely abolished by A23187 when cells were stimulated for 2 h. The inhibitory signal induced by A23187 was completely prevented by calpain, but not by caspase, inhibitors, suggesting a role for these proteases (Fig. 3B and data not shown). This hypothesis was supported by the loss of STAT6 protein in IL-4-stimulated cells treated with A23187 for 2 h (Fig. 3B). In these cells the amount of total STAT6 protein was lower than that in untreated cells or in cells treated with IL-4 or A23187 alone. The degradation of STAT6 under these conditions was also prevented by the calpain inhibitor ALLN. Because the loss of STAT6 protein correlated with the inhibition of phosphorylation, it is possible that only phosphorylated STAT6 was an in vivo substrate for calpains. The degradation of STAT6 was dependent on the length of cell stimulation. The need for a longer period of stimulation may suggest that de novo protein synthesis was required for the degradation as is seen in the inhibition of the JAK/STAT pathway by suppressor of cytokine signaling (17, 19). However, the protein synthesis inhibitor cycloheximide did not prevent the loss of phosphorylated and total STAT6 protein induced by A23187 in IL-4-stimulated cells (Fig. 3C).
FIGURE 3. Calcium signaling induces the loss of phosphorylated STAT6. A, A1.1 cells were left untreated or were stimulated with 1 μM of the calcium ionophore A23187 in the presence or the absence of ALLN. After 30 min of incubation at 37°C, STAT6 was immunoprecipitated and analyzed by SDS-PAGE with anti-STAT6 Ab. B, A1.1 cells were first incubated with the calpain inhibitor ALLN for 10 min before the addition of A23187 (Io) for an additional 10 min. Then IL-4 (10 ng/ml) was added, and cells were incubated for 30 min (left) and 2 h (right). As controls, cells were left untreated (Untreat.) or were stimulated with A23187 alone (Io). After incubation, STAT6 was immunoprecipitated from cell lysates. Immunoprecipitates were separated by SDS-PAGE and immunoblotted with an antiphosphotyrosine Ab to detect tyrosine-phosphorylated STAT6 (Py-STAT6; upper blots). Membranes were reprobed with anti-STAT6 Ab (STAT6; lower blots). The graphics show the relative densities of the STAT6 bands in the total STAT6 protein blots. C, A.1.1 cells were treated with ALLN for 10 min before the addition of A23187 (Io) or A23187 (Io) plus 25 μg/ml cycloheximide (CHX) for 15 min. Then IL-4 was added where indicated, and cells were incubated for an additional 2 h. After that, STAT6 was immunoprecipitated and analyzed as described in B. These experiments were performed at least three times.
STAT6 degradation by cytoplasmic calpains
Calpains are mainly cytosolic proteases, although they can be found in the nucleus (26, 28). Because STAT6 protease activity has been described in the nucleus (20, 21), we investigated the cell compartment in which STAT6 was proteolytically processed. As a first approach, STAT6 was analyzed in cytoplasmic and nuclear extracts from cells stimulated with nothing, IL-4, A23187, or IL-4 plus A23187 (Fig. 4). The stimulation of A1.1 cells with IL-4 for 30 min provoked the phosphorylation and migration of STAT6 to the nuclear compartment as previously reported (17, 18). At this time, STAT6 was not found in the nucleus of unstimulated cells. However, a large amount of STAT6 protein was found in the nuclear compartment of IL-4-stimulated cells (Fig. 4A). Treatment of A.1.1 cells with A23187 for 30 min did not affect the regulation of STAT6 by IL-4. The phosphorylation levels of STAT6 diminished after 2 h of stimulation with IL-4 compared with 30 min (Fig. 4B). In this case, phosphorylated STAT6 was also found in the nucleus after IL-4 stimulation. As expected, treatment of IL-4-stimulated cells with A23187 for 2 h resulted in the loss of phosphorylated STAT6 in the cytoplasmic and nuclear stores. This effect was prevented by treatment of cells with ALLN. When total STAT6 protein was analyzed, its cytoplasmic levels were slightly lower in A1.1 cells treated with IL-4 than in its absence. This correlated with the appearance of STAT6 in the nucleus of IL-4-stimulated cells (Fig. 4B). In contrast to that at 30 min, the amount of total STAT6 protein was higher in the cytosol than in the nucleus of IL-4-stimulated cells, suggesting that dephosphorylated STAT6 could have returned to the cytoplasm as described by others (17, 18). Interestingly, treatment of IL-4-stimulated cells with the calcium ionophore A23187 for 2 h did not alter the expression of STAT6 protein in the cytosol (Fig. 4B). In contrast, STAT6 was completely absent in the nucleus of IL-4-stimulated cells treated with A23187. This was also prevented by the calpain inhibitor ALLN. Therefore, the loss of STAT6 protein induced by calcium ionophore correlated with the loss of phosphorylated nuclear STAT6, suggesting that only phosphorylated STAT6 was an in vivo substrate for calpains.
FIGURE 4. Analysis of STAT6 degradation in cellular compartments. A1.1 cells were incubated with ALLN before the addition of A23187 (Io) for 10 min. Then cells were stimulated, or not, with IL-4 for 30 min (A) and 2 h (B). Cytoplasmic and nuclear extracts were prepared as described in Materials and Methods. STAT6 was immunoprecipitated from these extracts, and its phosphorylation (Py-STAT6; upper blots) and total protein (STAT6; lower blots) were determined as indicated in Fig. 3. As a control, cells were cultured in the absence of stimulus (Untreat.). The graphics show the relative densities of the STAT6 bands in the total STAT6 protein blots. These experiments were performed at least three times.
The data reported above describe the absence of nuclear STAT6 protein in IL-4-stimulated cells treated with A23187. However, it was not determined in what cell compartment STAT6 was proteolytically processed. It could be that phosphorylated STAT6 was degraded by a nuclear protease. Alternatively, it is possible that a cytoplasmic protease degrades phosphorylated STAT6, preventing its migration to the nucleus. To test these possibilities, the presence of calcium-dependent STAT6 proteases was analyzed in isolated cytoplasmic and nuclear extracts (Fig. 5). To this end, phosphorylated STAT6 was isolated by immunoprecipitation from IL-4-stimulated cells. Then, it was incubated with cytoplasmic and nuclear extracts prepared from A1.1 cells stimulated with A23187 for 2 h. Thus, phosphorylated STAT6 was completely degraded when incubated with cytoplasmic extracts in the presence of calcium. This effect correlated with the loss of total STAT6 protein and was prevented by the calpain inhibitor ALLN. In contrast, STAT6 protein levels and phosphorylation were not affected by calcium after incubation with nuclear extracts. These data show that STAT6 can be proteolytically processed by cytoplasmic calpains.
FIGURE 5. Degradation of STAT6 by cytoplasmic calpains. A1.1 cells were stimulated with A23187 for 2 h before the extraction of cytoplasmic and nuclear extracts in the absence of protease inhibitors. In parallel, phosphorylated STAT6 was precipitated from IL-4-stimulated cells. Then this precipitated STAT6 was incubated with the cytoplasmic (left) and nuclear (right) extracts in the presence of ALLN for 10 min. After that, samples were supplemented, or not, with 3 mM CaCl2 and incubated for 20 min at 20°C. STAT6 was separated by SDS-PAGE and analyzed with anti-tyrosine Ab (Py-STAT6; upper blots), followed by anti-STAT6 Ab (STAT6; lower blots). As controls, samples were incubated in the absence of CaCl2 (untreat.). These experiments were performed at least three times.
Discussion
STAT6 is a transcription factor that plays an important role in cell responses to IL-4 (12). Its activation is tightly regulated by kinases and phosphatases (14, 15, 16, 17, 18). In the past few years, several reports indicated that proteases can also regulate STAT6 (17, 20, 21, 22). In mast cells, STAT6 can be cleaved to a shorter isoform by nuclear serine proteases (20, 21) and calpains (22). The proteasome by indirect mechanisms can also down-regulate activated STAT6 (17, 19). The data reporting proteolytic processing of STAT6 is mainly described in mast cells (20, 21, 22). In this study we investigated the nature of STAT6 proteases in T cell lines. The results suggested that signals that promote calcium mobilization can negatively regulate IL-4-mediated STAT6 activation by a calpain-dependent mechanism.
Our results indicate that calpains can target and completely degrade STAT6 by a calcium-dependent mechanism. These results are in contrast with a report showing that STAT6 was cleaved to a shorter 70-kDa form by calpain (22). Explanations for this difference may be due to variations in the experimental conditions used. The previous study used mast cell extracts. It is possible that mast cell extracts contain factors that would interfere with the activity of calpain on STAT6. The existence of cytoplasmic STAT6-binding factors can support this hypothesis (31). Moreover, it may be possible that different post-translational modifications of STAT6 in mast cells and T cells may influence the action of calpains. In our case, STAT6 was analyzed in a T cell line; furthermore, it was purified by immunoprecipitation, which could eliminate potential interfering factors. An analysis with the PEST-FIND program indicated that STAT6 contains several potential PEST domains susceptible to calpain cleavage (25). The presence of multiple calpain sites would be consistent with the disappearance of STAT6 protein when incubated with these proteases. Thus, the digestion of STAT6 by calpains would produce fragments too small to be detected by Western blot.
The effect of calpain on STAT6 appears to be more complex in vivo. Calpains are activated by signals that raise intracellular calcium, such as calcium ionophores and engagement of Ag receptors (27, 28). However, cell signaling with calcium ionophores was not enough to induce STAT6 degradation. One possible explanation is that STAT6 is somehow protected in vivo from calpain activity. In this regard, it has recently been reported that a cytoplasmic factor can be bound to STAT6 (31). It could be possible that a similar factor would protect STAT6 from calpain in cells. The preparation of cell extracts with detergent would remove this factor, allowing STAT6 interaction with calpain in vitro.
The data suggested that only phosphorylated STAT6 was degraded by calpains in intact cells. Thus, treatment of IL-4-stimulated cells with calcium ionophore resulted in the absence of phosphorylated STAT6. This inhibition was blocked by calpain inhibitors, but not by caspase and serine proteases, indicating a role for calpain proteases in the regulation of phosphorylated STAT6 in vivo. Furthermore, the calcium-dependent degradation of total STAT6 protein only occurred when cells were stimulated with IL-4 (Figs. 3 and 4). It could be possible that the phosphorylation of STAT6 by IL-4 would allow its interaction with calpain. In this regard, tyrosine phosphorylation has been shown to facilitate protein cleavage by calpains (29, 30). The fact that the maximum inhibitory effect was observed after 2 h of stimulation also suggests that other regulatory proteins were involved. The mechanisms are still under investigation, but they do not implicate de novo protein synthesis, because cycloheximide did not prevent the effect of ionophores on STAT6. Interestingly, the cleavage of ZAP-70 by calpain is similar to that of STAT6 (29). In that case, calcium signaling was also not sufficient to promote ZAP-70 cleavage; the activation of ZAP-70 tyrosine kinases was required. It is possible that ZAP-70 and STAT6 share a similar mechanism of degradation by calpains.
Calpains have been described in T cells, and their expression and activity are increased after TCR engagement (27). Calcium mobilization signals have been proposed to regulate T cell differentiation (32, 33). The stimulation of unprimed T cells with strong calcium signaling can favor the development of Th1 cells (32), and after T cell stimulation, the increase in intracellular calcium is lower in Th2 cells (33). It is possible that the activation of calpains by signals that promote strong calcium signaling would regulate T cell differentiation through degradation of STAT6.
The data reported in this study indicate that calpains can down-regulate activated STAT6. Several findings support this conclusion: 1) STAT6 is degraded by calpains; 2) calcium signaling provoked the loss of phosphorylated STAT6; 3) the lack of phosphorylated STAT6 correlated with the loss of nuclear protein; and 4) these effects were prevented by calpain inhibitors. These findings reveal a novel mechanism of STAT6 regulation. Thus, calcium-activated calpains can act to degrade tyrosine-phosphorylated STAT6 in the cytoplasm, thus preventing its migration into the nucleus.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Dr. Achsah D. Keegan for helpful reading of the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported in part by Fondo de Investigacion Sanitaria Grant 01/0157 and Junta de Extremadura Grant 2PR01C015. M.P.G. is supported by the Fundacion Fernando Valhondo Calaff, and J.Z. is supported by the Subdireccion General de Investigacion Sanitaria, Exp. 99/3082.
2 Address correspondence and reprint requests to Dr. Jose Zamorano, Unidad de Investigacion, Hospital San Pedro de Alcantara. Avenida Pablo Naranjo s/n, 10003 Caceres, Spain. E-mail address: jose.zamorano{at}ses.juntaex.es
3 Abbreviations used in this paper: ALLN, N-acetyl-Leu-Leu-Nle-CHO; PEST, proline, glutamic acid, serine, threonine; z-VAD, z-Val-Ala-Asp-fluoromethylketone.
Received for publication February 27, 2004. Accepted for publication December 15, 2004.
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The transcription factor STAT6 plays an important role in cell responses to IL-4. Its activation is tightly regulated. STAT6 phosphorylation is associated with JAKs, whereas dephosphorylation is associated with specific phosphatases. Several studies indicate that proteases can also regulate STAT6. The aim of this study was to investigate the nature of these proteases in mouse T cell lines. We found that STAT6 was degraded in cell extracts by calcium-dependent proteases. This degradation was specifically prevented by calpain inhibitors, suggesting that STAT6 was a target for these proteases. This was supported by the cleavage of STAT6 by recombinant calpains. The proteolytic regulation of STAT6 was more complex in vivo. Calcium signaling was not sufficient to induce STAT6 degradation. However, treatment of IL-4-stimulated cells with calcium ionophores resulted in the absence of phosphorylated STAT6. This effect correlated with the loss of STAT6 protein and was prevented by calpain inhibitors. Cytoplasmic calpains seemed to be responsible for STAT6 degradation. Calpains can target signaling proteins; in this study we found that they can negatively regulate activated STAT6.
Introduction
Interleukin-4 is a cytokine with a wide spectrum of biological activities (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). IL-4 is mainly produced by T cells, mast cells, basophils, and eosinophils, but its effects are not limited to the immune system (2). IL-4 can regulate proliferation, differentiation, and apoptosis in multiple cell types of hemopoietic and nonhemopoietic origin (3, 4, 5). One of the most important effects of IL-4 is the regulation of T cell differentiation. IL-4 promotes the formation of Th2 cells while inhibiting Th1 (6). IL-4-driven Th2 cells can be protective in rheumatoid arthritis (7) and helminthic infections (8), but conversely, they can have an important role in allergic disease progression (9).
Responses to IL-4 are mediated by a cell surface receptor complex expressed in most cell types (2). This receptor consists of two subunits, the IL-4R -chain (IL-4R) and the common -chain (10). The binding of IL-4 to its receptor provokes the activation of JAK tyrosine kinases and the phosphorylation of tyrosine residues within the cytoplasmic tail of the receptor (11). Thus, domains of the IL-4R containing phosphorylated tyrosines can recruit intracellular signaling molecules. Among them, the transcription factor STAT6 plays an important role, as demonstrated by the similar phenotype of mice lacking the IL-4R and those lacking STAT6 (12).
The mechanism proposed for STAT6 activation is similar to that of other STATs (11, 12, 13, 14, 15). STAT6 binds through its Src homology 2 domain to specific phosphotyrosine residues within the IL-4R (15). In this complex, STAT6 is also phosphorylated at tyrosine residues (14). Subsequently, STAT6 leaves the receptor, dimerizes, and migrates to the nucleus, where it binds to consensus sequences in the promoter of genes (2, 13). It is believed that STAT6 is tightly regulated by kinases, phosphatases, and proteases. Since its discovery, the activation of STAT6 was associated with JAKs (11, 14). However, other tyrosine kinases, such as Src, can participate in the earlier events that lead to STAT6 activation (16). The inhibition of STAT6 is also strongly regulated, because in the absence of IL-4, STAT6 is quickly deactivated. Thus, phosphatases, by dephosphorylating STAT6 (17, 18), and suppressor of cytokine signaling, by blocking the kinase activity associated with the receptor (19), are inhibitors of STAT6.
Recent findings indicate that proteases can also be involved in the regulation of STAT6 (17, 20, 21, 22). Two research groups have reported that the cleavage of STAT6 by nuclear proteases can be an important event in the regulation of transcription in mast cells (20, 21). The nature of these proteases is still under investigation, although the data reported suggest that they may belong to the family of serine proteases. Calpains have been found to be able to cleave STAT6 (22) and other STATs (23), although their role in signaling is still unknown. The proteasome has also been involved in the inhibition of STAT6, although by an indirect mechanism (17, 19). Given the importance that proteases may have in the regulation of STATs, we investigated the nature of STAT6 proteases and their role in signaling. We found that in vitro, STAT6 was proteolytically degraded by calpains in a calcium-dependent manner. However, calcium signaling was not enough to promote in vivo STAT6 degradation. Our results suggest that only IL-4-activated STAT6 was an in vivo substrate for calpain proteases.
Materials and Methods
TCells and reagents
The murine T cell hybridoma A1.1 was maintained in RPMI 1640 culture medium with glutamine, penicillin, streptomycin, and 10% FCS (complete medium). A23187, N-acetyl-Leu-Leu-Nle-CHO (ALLN),3 N-acetyl-Leu-Leu-Met-CHO, calpastatin peptide, recombinant rat m-calpain, and purified μ-calpain from human erythrocytes were purchased from Calbiochem. The caspase inhibitors Boc-Asp-fluoromethylketone and z-Val-Ala-Asp-fluoromethylketone (z-VAD) were obtained from Alexis. RC20 anti-phosphotyrosine Ab was purchased from Transduction Laboratories, and M20 and M200 anti-STAT6 Abs were obtained from Santa Cruz Biotechnology. Cytokines were purchased from R&D Systems. The rest of the reagents used were purchased from Sigma-Aldrich.
Protease assays in cell extracts
After culture, cells were centrifuged and lysed with lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 0.5% Brij 35, and 0.1% Nonidet P-40) without inhibitors. Lysates were then clarified by centrifugation at 20,000 x g for 10 min. The soluble fraction was incubated as indicated to determine STAT6 protease activity. The reaction was stopped by adding 2x SDS sample buffer, and samples were separated on a 7.5% SDS-polyacrylamide gel before transfer to a polyvinylidene difluoride membrane. Membranes were then probed with M20 or M200 anti-STAT6 Abs. The bound Ab was detected using ECL (Pierce).
Immunoprecipitation and immunoblotting
These experiments were performed as we have previously described (16). After the indicated culture conditions, cell pellets were lysed with lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 50 mM sodium fluoride, 10 mM pyrophosphate, 1 mM PMSF, and protease inhibitor mixture) and clarified by centrifugation. The soluble fraction was incubated with anti-STAT6 Ab, followed by protein G-agarose. The washed precipitates were analyzed by Western blot as described above.
Preparation of cytoplasmic and nuclear extracts
Nuclear and cytoplasmic extracts were obtained as previously described with some modification (18, 24). After culture, cells were washed with buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 1,5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, and protease inhibitor mixture). Then cells were incubated for 20 min on ice with buffer A supplemented with 0.1% Nonidet P-40. Samples were centrifuged at 14,000 x g for 30 s. The supernatant containing the cytoplasmic extract was retained. The pellet containing nuclei was washed twice with buffer A, then resuspended in buffer B (20 mM HEPES (pH 7.9), 420 mM NaCl, 1,5 mM MgCl2, 1 mM EDTA, 0.5% Nonidet P-40, 0.5 mM DTT, and protease inhibitor mixture). After 10 min of incubation, samples were centrifuged at 14,000 x g for 10 min. The supernatant containing the nuclear extract was diluted with buffer B without NaCl to a final concentration of 150 mM NaCl. STAT6 was then precipitated and analyzed in cytoplasmic and nuclear extracts as described above.
To investigate STAT6 protease activity, cytoplasmic and nuclear extracts were obtained from A23187-stimulated cells as described above, but all procedures were performed in the absence of EDTA and protease inhibitors. In parallel, STAT6 was precipitated from IL-4-stimulated cells, and immunocomplexes were extensively washed in protease reaction buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 0.5% Brij 35, 0.1% Nonidet P-40, and 0.5 mM DTT). Then immunoprecipitates containing STAT6 were incubated with cytoplasmic and nuclear extracts in reaction buffer. After that, immunoprecipitates containing STAT6 were analyzed by Western blot as described above. The relative densities of STAT6 bands were analyzed in the total STAT6 protein blots using Science Lab software from Fuji Photo Film.
Results
Degradation of STAT6 by calpains
Several groups have reported that proteases can participate in the regulation of STAT6, although the mechanisms are not fully understood (17, 20, 21, 22). In this study we investigated the nature of these proteases. As a first approach, we analyzed the stability of STAT6 in cell extracts obtained in the absence of protease inhibitors (Fig. 1A). Incubation of these extracts at 20 and 37°C resulted in no significant loss of STAT6 protein compared with samples kept on ice. The lack of STAT6 degradation could be due to the absence in the preparations of cofactors that are necessary for the enzymatic activity of proteases. Because cations are protease cofactors, we investigated whether they influenced the stability of STAT6 (Fig. 1B). The addition of Zn2+, Mg2+, and Mn2+ to the extracts had little effect on STAT6 protein levels. In contrast, the addition of Ca2+ provoked the complete loss of STAT6, indicating that it was a substrate for calcium-dependent proteases. To identify these proteases, these cell extracts were incubated with calcium in the presence of known protease inhibitors (Fig. 1C). Among all inhibitors tested, only those known to inhibit calpains were able to block the calcium-dependent STAT6 degradation. Thus, EDTA, ALLN, and calpastatin blocked the degradation of STAT6, whereas inhibitors of caspase (Boc-Asp-fluoromethylketone and z-VAD) and serine protease (N-p-tosyl-L-phenylalanine chloromethylketone) had no effect (Fig. 1C). These data strongly suggested that STAT6 was an in vitro substrate for calpain proteases. This was confirmed by the fact that STAT6 was cleaved by recombinant m-calpain (Fig. 2). To perform these experiments, STAT6 was immunoprecipitated from untreated A1.1 cells to avoid potential interfering factors that could be present in the cellular extracts. Thus, the incubation of precipitated STAT6 with recombinant m-calpain resulted in the degradation of STAT6 by a calcium-dependent mechanism (Fig. 2A, left). This effect was also prevented by calpain inhibitors. Similar results were
found when immunoprecipitated STAT6 was incubated with purified human m-calpain (Fig. 2A, right). To detect STAT6, we used the M200 anti-STAT6 Ab that recognizes an internal domain and can detect shorter products of STAT6 proteolysis (22). However, we did not observe shorter STAT6 fragments even though we carefully performed dose- and time-response experiments (Fig. 2B). Thus, immunoprecipitated STAT6 was quickly degraded by different doses of recombinant m-calpain in the presence of calcium. The loss of STAT6 was not accompanied by the appearance of shorter fragments. It could be possible that the cleavage of STAT6 by calpains would produce fragments too small to be detected. These findings were consistent with the presence of multiple potential PEST domains in STAT6 that can be cleaved by calpains (25). Similar results were obtained with M20 Ab that recognizes a STAT6 C-terminal domain (data not shown). Taken together, these data indicated that STAT6 was a substrate to be proteolytically degraded by calpain in vitro.
FIGURE 1. STAT6 is proteolytically degraded by calcium-dependent proteases. A, Cells extracts obtained from A1.1 cells in the absence of protease inhibitors were kept at 4°C or incubated at 20 and 37°C for 30 min. Then samples were separated by SDS-PAGE and immunoblotted with M200 anti-STAT6 Ab. B, Cell extracts were supplemented with 3 mM of the indicated cation and incubated for 30 min at 20°C. STAT6 was analyzed as described above. C, Cell extracts were left untreated (Untreat.) or were incubated for 10 min with nothing or with 10 mM EDTA, 5 μM calpastatin (CAST), 20 μM of the calpain inhibitors N-acetyl-Leu-Leu-Met-CHO and ALLN, 20 μM of the caspase inhibitor z-VAD, and 40 μM of the serine protease inhibitor N-p-tosyl-L-phenylalanine chloromethylketone. Then 3 mM CaCl2 was added, and samples were incubated for 20 min at 20°C. STAT6 was analyzed as described in A. These experiments were performed at least three times.
FIGURE 2. Degradation of STAT6 by recombinant calpains. A, STAT6 was immunoprecipitated from unstimulated A.1.1 cells. Then it was incubated with recombinant m-calpain (left) and purified human μ-calpain (right) for 10 min in the presence of nothing, EDTA, or the indicated protease inhibitors. To start the reactions, 3 mM CaCl2 was added, and samples were incubated for 20 min at 20°C. As a control, STAT6 was incubated with calpain in the absence of CaCl2 (NoCa). STAT6 was detected by Western blotting with M200 anti-STAT6 Ab. B, Immunoprecipitated STAT6 was incubated with the indicated amount of recombinant m-calpain before the addition of 3 mM CaCl2. Then samples were incubated for 2 and 4 min at 20°C. As controls, samples were kept on ice for 4 min (untreat). STAT6 was analyzed as described above. These experiments were performed at least three times.
Proteolytic regulation of STAT6 by calcium signaling
Calpains are calcium-dependent proteases that can cleave intracellular signaling proteins (26). Calcium ionophores increase intracellular calcium levels, provoking the activation of calpains (27, 28). However, stimulation of cells with the calcium ionophore A23187 did not induce STAT6 degradation in cells (Fig. 3A), suggesting that STAT6 could not be an in vivo calpain substrate. Alternatively, it could be possible that calcium signaling was not enough to promote STAT6 degradation. This hypothesis was based on the fact that some substrates require tyrosine phosphorylation to be cleaved by calpains (29, 30). Therefore, we investigated whether STAT6 phosphorylation by IL-4 could favor its degradation by calpain (Fig. 3B). IL-4 induced a rapid and strong phosphorylation of STAT6 that decayed over time, as previously demonstrated (17, 18). Thus, the levels of STAT6 phosphorylation were higher after 30 min than after 2 h of stimulation with IL-4. Treatment of A1.1 cells with calcium ionophore for 30 min did not
affect the ability of IL-4 to induce STAT6 phosphorylation. At this time, the levels of STAT6 protein also remained similar under all treatments. In contrast to that at 30 min, the phosphorylation of STAT6 induced by IL-4 was completely abolished by A23187 when cells were stimulated for 2 h. The inhibitory signal induced by A23187 was completely prevented by calpain, but not by caspase, inhibitors, suggesting a role for these proteases (Fig. 3B and data not shown). This hypothesis was supported by the loss of STAT6 protein in IL-4-stimulated cells treated with A23187 for 2 h (Fig. 3B). In these cells the amount of total STAT6 protein was lower than that in untreated cells or in cells treated with IL-4 or A23187 alone. The degradation of STAT6 under these conditions was also prevented by the calpain inhibitor ALLN. Because the loss of STAT6 protein correlated with the inhibition of phosphorylation, it is possible that only phosphorylated STAT6 was an in vivo substrate for calpains. The degradation of STAT6 was dependent on the length of cell stimulation. The need for a longer period of stimulation may suggest that de novo protein synthesis was required for the degradation as is seen in the inhibition of the JAK/STAT pathway by suppressor of cytokine signaling (17, 19). However, the protein synthesis inhibitor cycloheximide did not prevent the loss of phosphorylated and total STAT6 protein induced by A23187 in IL-4-stimulated cells (Fig. 3C).
FIGURE 3. Calcium signaling induces the loss of phosphorylated STAT6. A, A1.1 cells were left untreated or were stimulated with 1 μM of the calcium ionophore A23187 in the presence or the absence of ALLN. After 30 min of incubation at 37°C, STAT6 was immunoprecipitated and analyzed by SDS-PAGE with anti-STAT6 Ab. B, A1.1 cells were first incubated with the calpain inhibitor ALLN for 10 min before the addition of A23187 (Io) for an additional 10 min. Then IL-4 (10 ng/ml) was added, and cells were incubated for 30 min (left) and 2 h (right). As controls, cells were left untreated (Untreat.) or were stimulated with A23187 alone (Io). After incubation, STAT6 was immunoprecipitated from cell lysates. Immunoprecipitates were separated by SDS-PAGE and immunoblotted with an antiphosphotyrosine Ab to detect tyrosine-phosphorylated STAT6 (Py-STAT6; upper blots). Membranes were reprobed with anti-STAT6 Ab (STAT6; lower blots). The graphics show the relative densities of the STAT6 bands in the total STAT6 protein blots. C, A.1.1 cells were treated with ALLN for 10 min before the addition of A23187 (Io) or A23187 (Io) plus 25 μg/ml cycloheximide (CHX) for 15 min. Then IL-4 was added where indicated, and cells were incubated for an additional 2 h. After that, STAT6 was immunoprecipitated and analyzed as described in B. These experiments were performed at least three times.
STAT6 degradation by cytoplasmic calpains
Calpains are mainly cytosolic proteases, although they can be found in the nucleus (26, 28). Because STAT6 protease activity has been described in the nucleus (20, 21), we investigated the cell compartment in which STAT6 was proteolytically processed. As a first approach, STAT6 was analyzed in cytoplasmic and nuclear extracts from cells stimulated with nothing, IL-4, A23187, or IL-4 plus A23187 (Fig. 4). The stimulation of A1.1 cells with IL-4 for 30 min provoked the phosphorylation and migration of STAT6 to the nuclear compartment as previously reported (17, 18). At this time, STAT6 was not found in the nucleus of unstimulated cells. However, a large amount of STAT6 protein was found in the nuclear compartment of IL-4-stimulated cells (Fig. 4A). Treatment of A.1.1 cells with A23187 for 30 min did not affect the regulation of STAT6 by IL-4. The phosphorylation levels of STAT6 diminished after 2 h of stimulation with IL-4 compared with 30 min (Fig. 4B). In this case, phosphorylated STAT6 was also found in the nucleus after IL-4 stimulation. As expected, treatment of IL-4-stimulated cells with A23187 for 2 h resulted in the loss of phosphorylated STAT6 in the cytoplasmic and nuclear stores. This effect was prevented by treatment of cells with ALLN. When total STAT6 protein was analyzed, its cytoplasmic levels were slightly lower in A1.1 cells treated with IL-4 than in its absence. This correlated with the appearance of STAT6 in the nucleus of IL-4-stimulated cells (Fig. 4B). In contrast to that at 30 min, the amount of total STAT6 protein was higher in the cytosol than in the nucleus of IL-4-stimulated cells, suggesting that dephosphorylated STAT6 could have returned to the cytoplasm as described by others (17, 18). Interestingly, treatment of IL-4-stimulated cells with the calcium ionophore A23187 for 2 h did not alter the expression of STAT6 protein in the cytosol (Fig. 4B). In contrast, STAT6 was completely absent in the nucleus of IL-4-stimulated cells treated with A23187. This was also prevented by the calpain inhibitor ALLN. Therefore, the loss of STAT6 protein induced by calcium ionophore correlated with the loss of phosphorylated nuclear STAT6, suggesting that only phosphorylated STAT6 was an in vivo substrate for calpains.
FIGURE 4. Analysis of STAT6 degradation in cellular compartments. A1.1 cells were incubated with ALLN before the addition of A23187 (Io) for 10 min. Then cells were stimulated, or not, with IL-4 for 30 min (A) and 2 h (B). Cytoplasmic and nuclear extracts were prepared as described in Materials and Methods. STAT6 was immunoprecipitated from these extracts, and its phosphorylation (Py-STAT6; upper blots) and total protein (STAT6; lower blots) were determined as indicated in Fig. 3. As a control, cells were cultured in the absence of stimulus (Untreat.). The graphics show the relative densities of the STAT6 bands in the total STAT6 protein blots. These experiments were performed at least three times.
The data reported above describe the absence of nuclear STAT6 protein in IL-4-stimulated cells treated with A23187. However, it was not determined in what cell compartment STAT6 was proteolytically processed. It could be that phosphorylated STAT6 was degraded by a nuclear protease. Alternatively, it is possible that a cytoplasmic protease degrades phosphorylated STAT6, preventing its migration to the nucleus. To test these possibilities, the presence of calcium-dependent STAT6 proteases was analyzed in isolated cytoplasmic and nuclear extracts (Fig. 5). To this end, phosphorylated STAT6 was isolated by immunoprecipitation from IL-4-stimulated cells. Then, it was incubated with cytoplasmic and nuclear extracts prepared from A1.1 cells stimulated with A23187 for 2 h. Thus, phosphorylated STAT6 was completely degraded when incubated with cytoplasmic extracts in the presence of calcium. This effect correlated with the loss of total STAT6 protein and was prevented by the calpain inhibitor ALLN. In contrast, STAT6 protein levels and phosphorylation were not affected by calcium after incubation with nuclear extracts. These data show that STAT6 can be proteolytically processed by cytoplasmic calpains.
FIGURE 5. Degradation of STAT6 by cytoplasmic calpains. A1.1 cells were stimulated with A23187 for 2 h before the extraction of cytoplasmic and nuclear extracts in the absence of protease inhibitors. In parallel, phosphorylated STAT6 was precipitated from IL-4-stimulated cells. Then this precipitated STAT6 was incubated with the cytoplasmic (left) and nuclear (right) extracts in the presence of ALLN for 10 min. After that, samples were supplemented, or not, with 3 mM CaCl2 and incubated for 20 min at 20°C. STAT6 was separated by SDS-PAGE and analyzed with anti-tyrosine Ab (Py-STAT6; upper blots), followed by anti-STAT6 Ab (STAT6; lower blots). As controls, samples were incubated in the absence of CaCl2 (untreat.). These experiments were performed at least three times.
Discussion
STAT6 is a transcription factor that plays an important role in cell responses to IL-4 (12). Its activation is tightly regulated by kinases and phosphatases (14, 15, 16, 17, 18). In the past few years, several reports indicated that proteases can also regulate STAT6 (17, 20, 21, 22). In mast cells, STAT6 can be cleaved to a shorter isoform by nuclear serine proteases (20, 21) and calpains (22). The proteasome by indirect mechanisms can also down-regulate activated STAT6 (17, 19). The data reporting proteolytic processing of STAT6 is mainly described in mast cells (20, 21, 22). In this study we investigated the nature of STAT6 proteases in T cell lines. The results suggested that signals that promote calcium mobilization can negatively regulate IL-4-mediated STAT6 activation by a calpain-dependent mechanism.
Our results indicate that calpains can target and completely degrade STAT6 by a calcium-dependent mechanism. These results are in contrast with a report showing that STAT6 was cleaved to a shorter 70-kDa form by calpain (22). Explanations for this difference may be due to variations in the experimental conditions used. The previous study used mast cell extracts. It is possible that mast cell extracts contain factors that would interfere with the activity of calpain on STAT6. The existence of cytoplasmic STAT6-binding factors can support this hypothesis (31). Moreover, it may be possible that different post-translational modifications of STAT6 in mast cells and T cells may influence the action of calpains. In our case, STAT6 was analyzed in a T cell line; furthermore, it was purified by immunoprecipitation, which could eliminate potential interfering factors. An analysis with the PEST-FIND program indicated that STAT6 contains several potential PEST domains susceptible to calpain cleavage (25). The presence of multiple calpain sites would be consistent with the disappearance of STAT6 protein when incubated with these proteases. Thus, the digestion of STAT6 by calpains would produce fragments too small to be detected by Western blot.
The effect of calpain on STAT6 appears to be more complex in vivo. Calpains are activated by signals that raise intracellular calcium, such as calcium ionophores and engagement of Ag receptors (27, 28). However, cell signaling with calcium ionophores was not enough to induce STAT6 degradation. One possible explanation is that STAT6 is somehow protected in vivo from calpain activity. In this regard, it has recently been reported that a cytoplasmic factor can be bound to STAT6 (31). It could be possible that a similar factor would protect STAT6 from calpain in cells. The preparation of cell extracts with detergent would remove this factor, allowing STAT6 interaction with calpain in vitro.
The data suggested that only phosphorylated STAT6 was degraded by calpains in intact cells. Thus, treatment of IL-4-stimulated cells with calcium ionophore resulted in the absence of phosphorylated STAT6. This inhibition was blocked by calpain inhibitors, but not by caspase and serine proteases, indicating a role for calpain proteases in the regulation of phosphorylated STAT6 in vivo. Furthermore, the calcium-dependent degradation of total STAT6 protein only occurred when cells were stimulated with IL-4 (Figs. 3 and 4). It could be possible that the phosphorylation of STAT6 by IL-4 would allow its interaction with calpain. In this regard, tyrosine phosphorylation has been shown to facilitate protein cleavage by calpains (29, 30). The fact that the maximum inhibitory effect was observed after 2 h of stimulation also suggests that other regulatory proteins were involved. The mechanisms are still under investigation, but they do not implicate de novo protein synthesis, because cycloheximide did not prevent the effect of ionophores on STAT6. Interestingly, the cleavage of ZAP-70 by calpain is similar to that of STAT6 (29). In that case, calcium signaling was also not sufficient to promote ZAP-70 cleavage; the activation of ZAP-70 tyrosine kinases was required. It is possible that ZAP-70 and STAT6 share a similar mechanism of degradation by calpains.
Calpains have been described in T cells, and their expression and activity are increased after TCR engagement (27). Calcium mobilization signals have been proposed to regulate T cell differentiation (32, 33). The stimulation of unprimed T cells with strong calcium signaling can favor the development of Th1 cells (32), and after T cell stimulation, the increase in intracellular calcium is lower in Th2 cells (33). It is possible that the activation of calpains by signals that promote strong calcium signaling would regulate T cell differentiation through degradation of STAT6.
The data reported in this study indicate that calpains can down-regulate activated STAT6. Several findings support this conclusion: 1) STAT6 is degraded by calpains; 2) calcium signaling provoked the loss of phosphorylated STAT6; 3) the lack of phosphorylated STAT6 correlated with the loss of nuclear protein; and 4) these effects were prevented by calpain inhibitors. These findings reveal a novel mechanism of STAT6 regulation. Thus, calcium-activated calpains can act to degrade tyrosine-phosphorylated STAT6 in the cytoplasm, thus preventing its migration into the nucleus.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Dr. Achsah D. Keegan for helpful reading of the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported in part by Fondo de Investigacion Sanitaria Grant 01/0157 and Junta de Extremadura Grant 2PR01C015. M.P.G. is supported by the Fundacion Fernando Valhondo Calaff, and J.Z. is supported by the Subdireccion General de Investigacion Sanitaria, Exp. 99/3082.
2 Address correspondence and reprint requests to Dr. Jose Zamorano, Unidad de Investigacion, Hospital San Pedro de Alcantara. Avenida Pablo Naranjo s/n, 10003 Caceres, Spain. E-mail address: jose.zamorano{at}ses.juntaex.es
3 Abbreviations used in this paper: ALLN, N-acetyl-Leu-Leu-Nle-CHO; PEST, proline, glutamic acid, serine, threonine; z-VAD, z-Val-Ala-Asp-fluoromethylketone.
Received for publication February 27, 2004. Accepted for publication December 15, 2004.
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